Vaccine Design

Vaccine Design The Subunit and Adjuvant Approach

Pharmaceutical Biotechnology

Series Editor: Ronald T. Borchardt The University of Kansas Lawrence. Kansas

Volume 1 PROTEIN PHARMACOKINETICS AND METABOLISM Edited by Bobbe L. Ferraiolo, Marjorie A. Mohler, and Carol A. Gloff

Volume 2 STABILITY OF PROTEIN PHARMACEUTICALS, Part A: Chemical and Physical Pathways of Protein Degradation Edited by Tim J. Ahern and Mark C. Manning

Volume 3 STABILITY OF PROTEIN PHARMACEUTICALS, Part B: In Vivo Pathways of Degradation and Strategies for Protein Stabilization Edited by Tim 1. Ahern and Mark C. Manning

Volume 4 BIOLOGICAL BARRIERS TO PROTEIN DELIVERY Edited by Kenneth L. Audus and Thomas J. Raub

Volume 5 STABILITY AND CHARACTERIZATION OF PROTEIN AND PEPTIDE DRUGS: Case Histories Edited by Y. John Wang and Rodney Pearlman

Volume 6 VACCINE DESIGN: The Subunit and Adjuvant Approach Edited by Michael F. Powell and Mark J. Newman


Edited by

Michael F. Powell Genenteeh, [ne. South San Franeiseo, California

and

Mark J. Newman Vaxeel, [ne. Noreross, Georgia

Assistant Editor

J essica R. Burdman Genenteeh, [ne. South San Franciseo, California

With a Foreword by Jonas Salk

Volume 1

Springer Science+Business Media, LLC

Llbrary of Congress Cataloglng-ln-Publlcatlon Data

Vaccine design the subunit and adjuvant appraach I edited by Michael F. Powell and Mark J. Newman.

p. cm. -- (Pharmaceutical biatechnalagy; v. 6) Inc l udes b i b li agraph i ca l references and index. ISBN 978-1-4613-5737-7 ISBN 978-1-4615-1823-5 (eBook)

1. Vaccines. 2. Immunalagical adjuvants. 1. Pawell, Michael F. II. Newman, Mark J. III. Series. QR189.V24 1995 615' .372--dc20

ISBN 978-1-4613-5737-7

© 1995 Springer Science+Business Media New York Originally published by Plenum Press, New York in 1995 Soficover reprint of the hardcover 1 st edition 1995

109876543

AII rights reserved

95-16401 CIP

No part ofthis book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission from the Publisher

DOI 10.1007/978-1-4615-1823-5

To those people who have supported us with this effort and also throughout our careers. Mike gives special thanks to his

parents, Frank and Terri, in-laws, Sam and Joan, and especially to his wife Tana. Similarly, Mark thanks Joe, Adair, Kim,

and his wife Gale.

Contributors

Sally E. Adams • British Bio-technology Ltd., Oxford OX4 5LY, United Kingdom Jeff Alexander • Cytel Corporation, San Diego, California 92121 Alexander Andrianov • Virus Research Institute, Inc., Cambridge, Massachusetts

02138 Gail L. Barchfeld • Chiron Corporation, Emeryville, California 94608 Robert A. Baughman • Emisphere Technologies, Inc., Hawthorne, New York 10532 John W. Boslego • Walter Reed Army Institute of Research, Washington, D.C. 20307 Robert N. Brey • Vaxcel, Inc., Norcross, Georgia 30071 Carsten Brunn • Department of Pharmaceutics, University of Washington, School of

Pharmacy, Seattle, Washington 98195 Jeanine L. Bussiere • Department of Pathobiology and Toxicology, Genentech, Inc.,

South San Francisco, California 94080 Noelene E. Byars • Syntex Research, Palo Alto, California 94304 Esteban Celis • Cytel Corporation, San Diego, California 92121 Donna K.F. Chandler • Division of Vaccines and Related Products Applications, Center

for Biologics Evaluation and Research, Food and Drug Administration, Rockville, Maryland 20852

David Chernoff • Chiron Corporation, Emeryville, California 94608 Robert W. Chesnut • Cytel Corporation, San Diego, California 92121 Masatoshi Chi ba • Department of Chemical Engineering, Massachusetts Institute of Tech

nology, Cambridge, Massachusetts 02139; present address: Pharmaceutical Development Laboratory, Mitsubishi Kasei Corporation, Kashimi-gun, Ibaraki 314-02, Japan

Silas Chikunguwo • Department of Tropical Public Health, Harvard School of Public Health, Boston, Massachusetts 02115

Jeffrey L. Cleland • Pharmaceutical Research & Development, Genentech, Inc., South San Francisco, California 94080

Peter D. Cooper • Division of Cell Biology, John Curtin School of Medical Research, Australian National University, Canberra, ACT 2601, Australia

Richard T. Coughlin • Cambridge Biotech Corporation, Worcester, Massachusetts 01605

Daniel E. Cox • Cambridge Biotech Corporation, Worcester, Massachusetts 01605 Charles Dahl • Department of Biological Chemistry and Molecular Pharmacology,

Harvard Medical School, Boston, Massachusetts 02115 Lawrence W. Davenport • Genentech, Inc., South San Francisco, California 94080

vii

viii Contributors

Penny Dong • Department of Pharmaceutics, University of Washington, School of Pharmacy, Seattle, Washington 98195

Ronald Eby • Lederle-Praxis Biologicals, West Henrietta, New York 14586-9728 Ronald W. Ellis • Virus and Cell Biology, Merck Research Laboratories, West Point,

Pennsylvania 19486 Patricia E. Fast • Vaccine and Prevention Research Program, Division of AIDS, Na

tional Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892

Donald P. Francis • Genentech, Inc., South San Francisco, California 94080 Glenn R. Frank • Paravax, Inc., Fort Collins, Colorado 80526 Steve Furlong • Department of Rheumatology and Immunology, Harvard Medical

School, Boston, Massachusetts 02115 Reinhard Gltick • Department of Virology, Swiss Serum and Vaccine Institute Bern,

CH-300l Bern, Switzerland Karen L. Goldenthal • Division of Vaccines and Related Products Applications, Center

for Biologics Evaluation and Research, Food and Drug Administration, Rockville, Maryland 20852

Michael G. Goodman • Department of Immunology, The Scripps Research Institute, La Jolla, California 92037

Susan Gould-Fogerite • Department of Laboratory Medicine and Pathology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07103

James D. Green • Department ofPathobiology and Toxicology, Genentech, Inc., South San Francisco, California 94080

Howard M. Grey • Cytel Corporation, San Diego, California 92121 Robert B. Grieve • Paravax, Inc., Fort Collins, Colorado 80526 Rajesh K. Gupta • Massachusetts Public Health Biologic Laboratories, State Labora

tory Institute, Boston, Massachusetts 02130 Susan Haas • Emisphere Technologies, Inc., Hawthorne, New York 10532 Justin Hanes • Department of Chemical Engineering, Massachusetts Institute of Tech

nology, Cambridge, Massachusetts 02139 Donald A. Ham • Department of Tropical Public Health, Harvard School of Public

Health, and Department of Rheumatology and Immunology, Harvard Medical School, Boston, Massachusetts 02115

Mary Kate Hart • Division of Virology, U.S. Army Medical Research Institute for Infectious Diseases, Fort Detrick, Frederick, Maryland 21702

Barton F. Haynes • Department of Rheumatology and Immunology and the Duke Center for AIDS Research, Duke University Medical Center, Durham, North Carolina 27710

Andrew W. Heath • Department of Medical Microbiology, University of Sheffield Medical School, Sheffield S10 2RX, United Kingdom

Stanley L. Hem • Department ofIndustrial and Physical Pharmacy, Purdue University, West Lafayette, Indiana 47907

Rodney J.Y. Ho • Department of Pharmaceutics, University of Washington, School of Pharmacy, Seattle, Washington 98195

Contributors ix

Stephen L. Hoffman • Malaria Program, Naval Medical Research Institute, Bethesda, Maryland 20889-5055

Glenn Ishioka • Cytel Corporation, San Diego, California 92121

Sharon A. Jenkins • Virus Research Institute, Inc., Cambridge, Massachusetts 02138 Charlotte Read Kensil • Cambridge Biotech Corporation, Worcester, Massachusetts

01605 Alan 1. Kingsman • British Bio-technology Ltd., Oxford OX4 5LY, and Department of

Biochemistry, Oxford University, Oxford OXI 3QU, United Kingdom

Peter 1. Kniskern • Virus and Cell Biology, Merck Research Laboratories, West Point, Pennsylvania 19486

JOrg Kreuter • Institut fur Pharmazeutische Technologie, Johann Wolfgang GoetheUniversitat, D-60439 Frankfurt am Main, Germany

Ralph T. Kubo • Cytel Corporation, San Diego, California 92121

Lawrence B. Lachman • Department of Cell Biology, University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030

John R. La Montagne • Division of Microbiology and Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892

Robert Langer • Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Dale N. Lawrence • Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892

Deborah M. Lidgate • Syntex Research, Palo Alto, California 94304 B. Michael Longenecker • Department of Immunology, Faculty of Medicine, Univer

sity of Alberta, Edmonton, and Biomira Inc., Edmonton, Alberta T6N IH1, Canada

Jianneng Ma • Cambridge Biotech Corporation, Worcester, Massachusetts 01605 Raphael J. Mannino • Department of Laboratory Medicine and Pathology, University

of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07103

Stephen Marburg • Synthetic Chemical Research, Merck Research Laboratories. Rahway, New Jersey 07065

George C. McCormick • Department ofPathobiology and Toxicology, Genentech, Inc., South San Francisco, California 94080

Kent R. Myers • Ribi ImmunoChem Research, Inc., Hamilton, Montana 59840 Bernardetta Nardelli • Medicine Department, Infectious Diseases Division, New York

University Medical Center, New York, New York 10016 Mark J. Newman • Vaxcel, Inc., Norcross, Georgia 30071 A. D. M. E. Osterhaus • Department of Virology, Erasmus University Rotterdam, 3000

DR Rotterdam, The Netherlands Gary Ott • Chiron Corporation, Emeryville, California 94608 Thomas J. Palker • Department of Rheumatology and Immunology and the Duke

Center for AIDS Research, Duke University Medical Center, Durham, North Carolina 27710

Lendon G. Payne • Virus Research Institute, Inc., Cambridge, Massachusetts 02138

x Contributors

Christopher Penney • Immunomodulator Research Project, BioChem Therapeutic, Inc., Laval, Quebec H7V 4A7, Canada

Patricia 1. Freda Pietrobon • Connaught Laboratories, Inc., Swiftwater, Pennsylvania 18370

Michael F. Powell • Genentech, Inc., South San Francisco, California 94080 Ramachandran Radhakrishnan • Chiron Corporation, Emeryville, California 94608 Xiao-Mei Rao • Department of Cell Biology, University of Texas M.D. Anderson

Cancer Center, Houston, Texas 77030 Edgar Relyveld • Office d'Etudes en Vaccinologie, Paris, France Sandra R. Reynolds • Department of Tropical Public Health, Harvard School of Public

Health, Boston, Massachusetts 02115 G. F. Rimmelzwaan • Department of Virology, Erasmus University Rotterdam, 3000

DR Rotterdam, The Netherlands Bryan E. Roberts • Virus Research Institute, Inc., Cambridge, Massachusetts 02138 Bradford E. Rost • Massachusetts Public Health Biologic Laboratories, State Labora

tory Institute, Boston, Massachusetts 02130 John B. Sacci, Jr. • Malaria Program, Naval Medical Research Institute, Bethesda, and

Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland 21201

John Samuel • Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta T6G 2N8, Canada

Noemi Santiago • Emisphere Technologies, Inc., Hawthorne, New York 10532 Leigh A. Sawyer • Vaccine and Prevention Research Program, Division of AIDS,

National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892

Alessandro Sette • Cytel Corporation, San Diego, California 92121 Li-Chen N. Shih • Department of Cell Biology, University of Texas M.D. Anderson

Cancer Center, Houston, Texas 77030 George R. Siber • Massachusetts Public Health Biologic Laboratories, State Labora

tory Institute, Boston, Massachusetts 02130 Laura L. Snyder • ImmunoTherapy Corporation, 14262 Franklin Avenue, Tustin, Cali

fornia 92680 Sean Soltysik • Cambridge Biotech Corporation, Worcester, Massachusetts 01605 Vernon C. Stevens • Department of Obstetrics and Gynecology, The Ohio State Uni

versity, Columbus, Ohio 43210 James P. Tam • Microbiology and Immunology Department, Vanderbilt University,

Nashville, Tennessee 37232 Pierre L. Triozzi • The Ohio State University Comprehensive Cancer Center, The

Arthur G. James Cancer Hospital and Research Institute, Columbus, Ohio 43210 Cynthia A. Tripp • Paravax, Inc., Fort Collins, Colorado 80526 Stephen E. Ullrich • Department of Immunology, University of Texas M.D. Anderson

Cancer Center, Houston, Texas 77030 J. Terry Ulrich • Ribi ImmunoChem Research, Inc., Hamilton, Montana 59840 Peter van Hoogevest • Ciba Geigy Ltd., Basel, Switzerland Gary Van Nest • Chiron Corporation, Emeryville, California 94608

Contributors xi

Antonella Vitiello • Cytel Corporation, San Diego, California 92121 Frederick R. Vogel • Division of AIDS, National Institute of Allergy and Infectious

Diseases, National Institutes of Health, Bethesda, Maryland 20892 Peggy Wentworth • Cytel Corporation, San Diego, California 92121 Susan L. Wescott • Vaccine and Prevention Research Program, Division of AIDS,

National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892

Joe L. White • Department of Agronomy, Purdue University, West Lafayette, Indiana 47907

Nancy Wisnewski • Paravax, Inc., Fort Collins, Colorado 80526 David V. Woo • ImmunoTherapy Corporation, Tustin, California 92680 Jia-Yan Wu • Cambridge Biotech Corporation, Worcester, Massachusetts 01605

Foreword

When my interest was first drawn to the phenomenon of vaccination for virus diseases in the late 1930s, the state of the art and the science of vaccine design was not far advanced beyond the time of Jenner at the end of the 18th century and of Pasteur a century later. In the 1930s it was still believed that for the induction of immunity to a virus-caused disease the experience of infection was required, but not for a toxin-caused disease such as diphtheria or tetanus, for which a chemically detoxified antigen was effective for immunization. This prompted the question as to whether it might be possible to produce a similar effect for virus diseases using nonreplicating antigens.

When in the 1930s and 1940s it was found possible to propagate influenza viruses in the chick embryo, protective effects could be induced without the need to experience infection by the use of a sufficient dose of a noninfectious influenza virus preparation. Later in the 1940s, it became possible to propagate polio and other viruses in cultures of human and monkey tissue and to immunize against other virus diseases in the same way. Later, with the advent of the era of molecular biology and genetic engineering, antigens and vaccines could be produced in new and creative ways, using either replicating or nonreplicating forms of the appropriate antigens for inducing a dose-related protective state.

Influenza virus vaccine effectiveness was observed to correlate with levels of serum antibody acting to block infection at the portal of entry. The need for antibody at the portal of entry in influenza virus infection is necessitated by the short incubation period (1 to 2 days), too short for an anamnestic antibody response to have a protective effect as in the prevention of paralysis following effective poliomyelitis vaccination. In poliomyelitis, the incubation period is sufficiently prolonged for the anamnestic response in a vaccinated individual to precede invasion of the central nervous system and prevent paralysis, and to prevent pharyngeal infection and spread of virus by that route.

The role and importance of the length of the incubation period and of the presence and persistence of immunological memory for long-term immunity to paralytic polio revealed that the presence of serum antibody at the time of exposure was not a necessary prerequisite for the prevention of paralysis; thus, the presence of immunological memory alone is the necessary and sufficient requirement for effectively preventing paralysis. To produce a persistent memory state a single dose of a sufficient concentration of antigen in saline can be uniformly effective when administered at any time after loss of maternal antibody. When incorporated in a mineral oil adjuvant as previously used for an influenza

xiii

xiv Foreword

virus vaccine, much smaller doses of antigen are effective even in the presence of maternal antibody.

It now appears that for the control of cell-associated or cell-free pathogens by immunoprophylaxis a Thl-type or Th2-type memory pattern, respectively, is required. This is now evident for the immunological control of HI V, as for other cell-associated pathogens, for which a Thl-type memory state is required, and for which many more adjuvants, as well as antigen preparations, are now available for enhancing the induction of either cell-mediated or antibody-mediated immunological memory, required to protect against either cell-associated or cell-free pathogens, respectively.

Thus, for the design of vaccines and vaccine strategies for the control of viruses and other microbial pathogens, consideration must be given to the requirements appropriate to influence the immunodynamics and pathodynamics for each pathogen for which a specific immunizing agent is intended. Since infectious agents for which humans are the only reservoir may be eradicated from the human population through the phenomenon of the herd effect, it is also necessary to consider the epidemio-dynamics as well.

Through the availability of an extensive armamentarium of reagents and the science to guide their use it will now be possible to design safe and effective vaccines and vaccine strategies, for use with a greater economy of means in suitable combinations to produce the desired effect for many more pathogens than can now be controlled by vaccination.

This comprehensive volume considers and elaborates on all the relevant considerations for vaccine design, revealing the many possibilities that now exist to meet the multiple challenges for the control of infection and disease that the science and technology of vaccinology now make possible.

JONAS SALK San Diego

Preface

Most of the more traditional types of vaccines developed for use in humans are based on replicating pathogens that have been attenuated. Vaccination with these agents results in a limited infection but with little or no disease pathogenesis and the resultant immune responses protect against the fully virulent pathogens. Attenuated vaccines are still widely used and efforts to develop the next generation continue. Subunit vaccines differ since they are based on only one or a few proteins or polysaccharides from the target pathogen. They can readily be designed for safety but their failure to induce the appropriate types of immune responses has plagued their development. Novel biotechnology techniques are now providing ways of producing better subunit vaccines and with these advances, we may soon be able to protect the population against many more diseases.

The purpose of this book is to provide descriptions of scientific theories, product development strategies, and results from preclinical studies for many of the newest subunit vaccines or components designed for use in subunit vaccine formulation, such as adjuvants and vaccine delivery systems. We have selected topics that we feel highlight many promising technologies and novel products rather than focusing on disease states or individual pathogens. Most of these products are still considered "experimental" and as such have only been evaluated in animals. However, the common goal within the scientific community is to contribute to the development of a product that will be evaluated in humans and hopefully licensed for use. We have therefore opened the book with several chapters describing immunological, formulation, public health, safety, regulatory, and clinical considerations relevant to the design and development of subunit vaccines. These topics are critical concerns for those who manufacture vaccines as well as for those who receive them.

Most prominent in this book are chapters describing new adjuvants and vaccine delivery systems. Our concentration on this area not only represents our personal biases but also illustrates the breadth of interest in these products. New adjuvants have potential for use with numerous vaccines, including some that are currently approved for human use. Many of the vaccine delivery systems are adjuvant active and also have potential for altering the mode of vaccine administration. For example, the use of new technologies may allow vaccines to be administered orally, rather than by needle injection, and controlled release systems may allow "single-shot" delivery, thus avoiding booster immunizations. Many of the adjuvant products described in this book have been or will soon be evaluated in human clinical trials and we fully believe our excitement will be justified.

xv

xvi Preface

Although new adjuvants will have an impact on the vaccine industry, they are useless without the appropriate vaccine immunogen. Here again, we have selected topics based on novel technologies for designing immunogens with potential for widespread application. We have also included products for many new applications for which commercially viable vaccines do not exist. Examples include the development of synthetic polysaccharideprotein conjugates and recombinant lipid-protein conjugates for use in vaccines against bacteria as well as recombinant protein and synthetic-peptide immunogens for use against parasites, viruses, cancer, and even pregnancy!

This field is evolving rapidly and, by definition, the information provided will not long remain current. We urge the interested readers to contact the authors directly with their scientific questions and suggestions. Finally, we have made every attempt to be comprehensive but it is impossible to include all of the subunit vaccine technologies under development. To the inventors and advocates of technologies that we may have missed, we apologize.

MARKJ. NEWMAN

Georgia MICHAEL F. POWELL

San Diego

Contents

List of Abbreviations ................................ XXXIX

Chapter 1

Immunological and Formulation Design Considerations for Subunit Vaccines Mark J. Newman and Michael F. Powell

1. Introduction ............................. . 2. Characterizations of Immune Responses Relevant to Vaccine Design

2.1. Adaptive and Innate Immunity . . . . . . . . . . . . . . . . 2.2. Antigen Processing and Presentation to the Immune System 2.3. Cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Immunological Memory .................. .

3. Immune Responses to Infectious Pathogens, Tumors, and Vaccines 3.1. Immune Responses against Extracellular Bacteria 3.2. Immune Responses against Intracellular Bacteria 3.3. Immune Responses to Viruses ....... . 3.4. Immune Responses against Parasites .... . 3.5. Nontraditional Uses of Vaccine Technologies

4. Design Criteria for Subunit Vaccines ...... . 4.1. Selection of Vaccine Immunogen. . . . . . . 4.2. The Use of Adjuvants to Alter Vaccine Immunogenicity . 4.3. ImmunizationlVaccination Schedule 4.4. Vaccine Delivery Route 4.5. Vaccine Formulation Stability . 4.6. Adjuvant Formulation Concerns

5. Conclusions References . . . . . . . . . . . . . .

Chapter 2 Public Health Implications of Emerging Vaccine Technologies Dale N. Lawrence, Karen L. Goldenthal, John W. Boslego, Donna K. F. Chandler; and John R. La Montagne

1. Introduction ...................... . 1.1. The Children's Vaccine Initiative ......... . 1.2. Unconquered Pathogens and Other Clinical Indications 1.3. Historical Model of Disease Eradication: Smallpox . . .

I 2 3 3 6 7 8 8

.10 · 12 · 14 · 16 · 17 · 17 · 18 · 21 .23 .23 .26 .27 .28

.43

.44

.44

.45

xvii

xviii Contents

1.4. Recent Model for Disease Control Approaching Eradication: Haemophilus inJluenzae Type b ............... . 45

2. Reevaluation of the Premises Underlying Public Health Vaccine Practices . 46 2.1. Premises Underlying Public Health Vaccine Programs and Practices . 46 2.2. New Technologies Altering the Public Health Vaccine Premises . 47

3. Public Health Impact of New Technologies . 51 3.1. Research and Development. . . . . . . . . . . . .51 3.2. Government Agency Activities . . . . . . . . . . . 52 3.3. Pri vate and Other Manufacturers of Vaccines and

Possible International Partnerships . . . . . . . . . 56 3.4. Opportunities for Scientific and Economic Advancement . 56

4. Summary. .57 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

Chapter 3 Preclinical Safety Assessment Considerations in Vaccine Development Jeanine L. Bussiere, George C. McCormick, and James D. Green 1. Introduction ..................... . 61 2. Potential Adverse Effects Associated with Vaccines . 62

2.1. General Toxicity of Vaccines . . . . 62 2.2. Enhancement of DiseaselInfection . 63 2.3. Antigenic Competition .. . . . 64 2.4. Unwanted Immune Responses . . . 65 2.5. Cross-Reactive Antibodies .. . . . 65 2.6. Teratogenic and Reproductive Effects . 67

3. Risks Experienced with Specific Types of Vaccines and Adjuvants . 67 3.1. Live Vaccines . . . . . 67 3.2. Killed Vaccines . . . . . . . . . . 68 3.3. Live Carrier Vaccines . . . . . . . 69 3.4. Genetically Engineered Vaccines . 70 3.5. Adjuvants . . . . . . . . . . . . . 71

4. Balancing Risks and Benefits . . . . . 72 5. Preclinical Safety Testing Considerations . 73

References . . . . . . . . . . . . . . . . . 75

Chapter 4 Regulatory Considerations in Vaccine Design Lawrence W Davenport 1. Introduction ................ . 2. Vaccine Development and Regulatory Issues 3. Center for Biologics Evaluation and Research 4. Code of Federal Regulations. . . . . . . . . . 5. Investigational New Drug Application .... 6. Product License and Establishment License Applications

. 81

.83

.89

.89

.90

.92

Contents

7. Guideline and Points to Consider Documents . 8. Summary .

References . . . . . . . . . . . . . . . . . . .

Chapter 5

xix

.94

.95

.96

Clinical Considerations in Vaccine Trials with Special Reference to Candidate HIV Vaccines Patricia E. Fast, Leigh A. Sawyer, and Susan L. Wescott 1. Introduction ................... . 97 2. Vaccine Candidates Currently in Clinical Trials. . . . . 98

2.1. Vaccine Candidates Other Than HIV-I . . . . . . 98 2.2. Types of HIV-I Vaccine Trials: Preventive, Therapeutic, and Perinatal 103 2.3. HIV-I Vaccine Candidates. . . . . . . . . . . . . . . 103 2.4. Sequential Use of Different Vaccines in One Regimen 104

3. Clinical Studies of Vaccine Candidates 107 3.1. Approach to Vaccine Trials . . . 107 3.2. Phases of Clinical Development 107

4. Factors in Design of Clinical Trials . . 110 4.1. Ethical Considerations in Testing HIV Vaccines 110 4.2. Volunteer Selection III 4.3. Special Populations . . . . . . . . . . . . . 112 4.4. Informed Consent . . . . . . . . . . . . . . 113 4.5. Age of Volunteers for HIV-l Vaccine Trials 114 4.6. Choice of Dose or Dose Range for Clinical Trials 114 4.7. Risks of Vaccination .... , .. .. .. .. .. . 115 4.8. Reimbursement .................. 117 4.9. Motivation of Volunteers in HIV-I Vaccine Trials 118 4.10. Randomization, Placebos, and Blinding. . . . . . ll8 4.11. Special Considerations for Vaccine Trials in Pregnant Women . 119 4.12. Special Considerations for Trials in Developing Countries. . . 120

5. Trial Outcome Measures. . . . . . . . . . . . . . . . . . . . . . . . 121 5.1. Laboratory Assays in HIV-l Prophylactic Trials: The Dilemma of

Unknown Correlate(s) ofImmunitylProtection . . . . . . . . . . . 121 5.2. Outcome Measures in HIV-l Therapeutic Trials: The Dilemma of

Unknown Surrogate Marker(s) 122 6. Conclusions 123

References . . . . . . . . . . . . . . 124

Chapter 6 Laboratory Empiricism, Clinical Design, and Social Value: The Rough Road toward Vaccine Development Donald P. Francis 1 . Introduction 2. Discussion . .

135 135

xx

3. Conclusion References

Chapter 7 A Compendium of Vaccine Adjuvants and Excipients Frederick R. Vogel and Michael F. Powell

Introduction .. . . . . . . . . Adju-Phos .......... . AF (see Antigen Formulation) Algal Glucan . Algammulin . . . . . . . . . . Alhydrogel . . . . . . . . . . . Alum (see Alhydrogel, Rehydragel) Aluminum Phosphate Gel (see Adju-Phos) Arlacel 85 (see Span 85) Antigen Formulation . Avridine® ....... . BAY Rl005 ...... . Block Copolymer (see CRL 1005) Calcitriol . . . . . . . . . . . . . . Calcium Phosphate Gel .. . . . . CFA (see Freund's Complete Adjuvant) Cholera Holotoxin (CT) . . . . . Cholera Toxin B Subunit (CTB) . CRLlO05 ............ . Cytokine-Containing Liposomes DDA .............. . Deoxycholic Acid (see DOC/Alum Complex) DHEA .................. . Dihydroxyvitamin D3 (see Calcitriol) Dimethylammonium Bromide (see DDA) DMPC ....... . DMPG ........... . DOC/Alum Complex ... . Freund's Complete Adjuvant Freund's Incomplete Adjuvant. Gamma Interferon (see Interferon-y) Gamma Inulin . Gerbu Adjuvant GM-CSF ... . GMDP .... . IFA (see Freund's Incomplete Adjuvant) IL-lf5 Peptide (see Sclavo Peptide) Imiquimod . ImmTher™ ............ .

Contents

139 139

141 142

143 145 146

147 148 149

150 151

152 152 154 156 157

158

159 160 161 162 163

164 165 166 167

168 169

Contents

Immune Stimulating Complex (see ISCOM) Interferon-y 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Interleukin-l p 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Interleukin-2 0 0

Interleukin-7 0 0

Interleukin-12 0

ISCOM(s)TM 0 0

Isoprep 7o0.3™ 0

Liposomes 0 0 0

Loxoribine 0 0 0

LT-OA or LT Oral Adjuvant MF59 0 0 0 000000000

Monophosphoryl Lipid A (see MPL®) Montanide® ISA 51 0 0

Montanide® ISA 720 0

MPL® 0000000

MTP-PE 0 0 0 0 0 0

MTP-PE Liposomes Murametide 0 0 0 0

Muramyl Dipeptide (see Threonyl-MDP) Murapalmitine 0 0

D-Murapalmitine 0 0 0 0 0 0 0 0

NAGO 0000000000000

Nonionic Surfactant Vesicles 0

Phosphatidylcholine (see DMPC) Phosphatidylglycerine (see DMPG) Pleuran 0 0 0 0 0 0 0 0 0

PLGAo PGA, and PLA 0

Pluronic Ll21 0

PMMA 0 0 0 000000

PODDSTM 0 00 0 0 0 0

Poloxamer 401 (see Pluronic Ll21) Polymethyl Methacrylate (see PMMA) Poly rA: Poly rU 0 0

Polyphosphazene 0 0

Polysorbate 80 0 0 0

Protein Cochleates 0

Proteinoid Microspheres (see PODDSTM) QS-21 0 0 0 0 0

QuilA 0 0 0 0 0

Rehydragel HPA Rehydragel LV 0

S-28463 0 0 0 0

SAF-I 0 0 0 0 0

Saponin (see Iscoprep, Quil A, QS-21)

xxi

170 171 172 174 175 177 178 179 181 182 183

184 185 186 188 189 190

191 192 193 195

196 198 200 201 202

203 204 205 206

208 210 211 212 213 214

xxii

Sclavo Peptide . . . . . . . . . . . Sendai Proteoliposomes . . . . . . Sendai-Containing Lipid Matrices. Sorbitan Trioleate (see Span 85) Span 85 ............ . Specol ............ . SPT (see Antigen Formulation) Squalane ... . Squalene ....... . Stearyl Tyrosine . . . . Stimulon™ (see QS-2) Termurtide™ (see Threonyl-MDP) Theramide™ ......... . Threonyl-MDP . . . . . . . . . Tween 80 (see Polysorbate 80) Ty Particles . . . . . . . . . . . . . . . . . Walter Reed Liposomes . . . . . . . . . . Acknowledgments and Update Summary .

Chapter 8 Adjuvant Properties of Aluminum and Calcium Compounds Rajesh K. Gupta, Bradford E. Rost, Edgar Relyveld, and George R. Siber 1. Introduction ....... . 2. Aluminum Compounds ...... .

2.1. Method of Preparation .... . 2.2. Factors Affecting the Adsorption 2.3. Adjuvant Properties . 2.4. Mechanism of Action 2.5. Limitations .... .

3. Calcium Phosphate ... . 3.1. Method of Preparation 3.2. Adjuvant Properties ..

4. Combination of Aluminum Compounds with Other Adjuvants. 5. Summary .

References

Chapter 9 Structure and Properties of Aluminum-Containing Adjuvants Stanley L. Hem and Joe L. White 1. Introduction ............ . 2. Characterization . . . . . . . . . . .

2.1. Aluminum Hydroxide Adjuvants 2.2. Aluminum Hydroxyphosphate Adjuvants 2.3. Commercial Vaccines . . . . . . . . .

3. Optimizing Antigen-Adjuvant Adsorption ..

Contents

215 216 216

218 219

220 221 222

223 224

225 226 227

229 230 230 231 233 237 238 239 239 241 241 242 242

249 249 251 253 257 258

Contents xxiii

3.1. Model Proteins .. . . . . . . . . . . . . . . 259 3.2. Malaria Antigens . . . . . . . . . . . . . . . 262

4. Effect of Protein Adsorption on Surface Properties 265 5. Interactions in Multivalent Vaccines Using Mixtures of Aluminum-Containing

Adjuvants ................ 267 5.1. Redistribution of Antigens .. . . . 267 5.2. Redistribution of Phosphate Anions 268 5.3. Colloidal Interactions . . . . . . 269

6. Effect of Anions . . . . . . . . . . . 270 6.1. Aluminum Hydroxide Adjuvant 271 6.2. Aluminum Phosphate Adjuvant. 272

7. Summary 274 References 275

Chapter 10 MF59: Design and Evaluation of a Safe and Potent Adjuvant for Human Vaccines Gary Ott, Gail L. Barchfeld, David Chernoff, Ramachandran Radhakrishnan, Peter van Hoogevest, and Gary Van Nest

1. Rationale for and Design of Microfluidized OillWater Emulsions 277 2. Preclinical Experience with MF59 . 281 3. Mechanism of Adjuvant Activity . . . . . . . . . . . 285 4. Manufacturing and Scaleup of MF59 . . . . . . . . . 288 5. Clinical Results with the MF59 Adjuvant Formulation 293 6. Summary . 294

References . . . . . . . . . . . . . . . . . . . . . . . 294

Chapter 11 Development of Vaccines Based on Formulations Containing Nonionic Block Copolymers Robert N. Brey

1. Introduction 2. Vaccine Delivery Systems and Adjuvants . . . . . . 3. Nonionic Block Polymers in Vaccine Formulations. 4. Properties of Vaccine Formulations Containing Nonionic Block Copolymers 5. Summary .

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Chapter 12 Development of an Emulsion-Based Muramyl Dipeptide Adjuvant Formulation for Vaccines Deborah M. Lidgate and Noelene E. Byars 1. Introduction ........ . 2. Compound Selection. . . . . 3. Chemistry of Threonyl-MDP

297 299 300 307 308 308

313 313 315

xxiv

4. Emulsion Fonnulation Considerations 5. SAF Fonnulation .. 6. SAF Manufacturing . 7. Final Product . . . . . 8. Mechanism of Action 9. Studies Utilizing SAF Emulsion with Threonyl-MDP

10. Conclusion References . . . . . . . . . . . . . . . . . . . . . . .

Chapter 13

Liposomal Presentation of Antigens for Human Vaccines

Reinhard Gliick 1. Introduction ..... . 2. History of Liposomes . 3. Liposomes as Adjuvants 4. Approaches with Liposomal Vaccines . 5. Technologies for Liposomal Vaccines . 6. Immunopotentiating Reconstituted Influenza Virosomes (lRIV) 7. Examples ofIRIV-Based Vaccines ...... . 8. Preclinical Evaluations ofIRIV-Based Vaccines ........ . 9. Clinical Evaluation ofIRIV-Based Vaccines .......... .

10. Patent Protection and Product Licensing of IRIV-Designed Vaccines 11. Prospects for the Future 12. Summary .

References

Chapter 14

Liposome Design and Vaccine Development

Patricia 1. Freda Pietrobon

Contents

315 316 317 319 321 321 322 322

325 326 327 327 329 331 335 336 336 339 340 340 341

1. Introduction ........................... 347 2. General Properties of Liposomes . . . . . . . . . . . . . . . . 348 3. Liposomes and Enhancement of Humoral Immune Responses . 349

3.1. Antibody Responses to Thymus-Independent Antigens . . 349 3.2. Liposome Stimulation of Thymus-Independent Type 1 and

Thymus-Independent Type 2 Antibody Responses. . . . . . 351 3.3. Liposomes That Provide T-Dependent Help to Weak (T-Independent)

Antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352 3.4. Liposomes and Enhancement of Thymus-Dependent Antibody Responses 353

4. Liposomes and Cell-Mediated Immune Responses . . . . . . . . . . . . . 354 4.1. Liposomal Antigen Delivery and CTL Induction. . . . . . . . . . . . 354 4.2. Liposomes and Their Effect on Antiviral and Antiparasitic Immunity. 355

5. Conclusion 356 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356

Contents

Chapter 15

Lipid Matrix-Based Vaccines for Mucosal and Systemic Immunization

Raphael 1. Mannino and Susan Gould-Fogerite 1. Introduction ....... . 2. Protein Cochleate Vaccines

2.1. Structure . . . . . . . . 2.2 Formulation .. . . . . 2.3. Oral Immunization and the Common Mucosal Immune System

xxv

363 364 365 366 367

2.4. Influenza: Immune Response to Cochleate Vaccines ...... 368 2.5. Parainfluenza Type 1: Immune Response to Cochleate Vaccines 372 2.6. Human Immunodeficiency Virus: Immune Response to Cochleate Vaccines 374

3. Fusogenic Proteoliposomes . . . . . . . 376 3.1. Introduction . . . . . . . . . . . . . . . . . . . 376 3.2. Sendai Fusogenic Proteoliposomes . . . . . . . 377

4. Unique Covalent Peptide-Phospholipid Complexes 379 4.1. Introduction . . . . . . . . . . . . . . . . . . . 379 4.2. Formulation . . . . . . . . . . . . . . . . . . . 380 4.3. Stimulation of an Immune Response to Hydrophilic B-CeJI Determinants

through an Association with Putative Th-Cell Determinants. 380 5. Summary . 382

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384

Chapter 16

Polymer Microspheres for Vaccine Delivery

Justin Hanes, Masatoshi Chiba, and Robert Langer 1. Introduction ................ . 2. Polymer Microspheres for Vaccine Delivery

2.1. Parenteral Immunization . . . . . . . 2.2. Mucosal Immunization . . . . . . . .

3. Polymers for Vaccine Microencapsulation 3.1. Lactide/Glycolide Polyesters ..... 3.2. Polymers with Built-in Adjuvanticity . 3.3. Gelatin. . . . . . . . . . . . . 3.4. Polyphosphazene Gels .... 3.5. Microencapsulated Liposomes

4. The Role of Immunostimulants .. 5. Microsphere Production ..... . 6. Consideration of Vaccine Formulation Stability. 7. Regulatory Issues 8. Conclusions

References . . . .

389 390 390 398 402 402 403 404 404 405 405 406 407 408 408 409

xxvi Contents

Chapter 17 Vehicles for Oral Immunization Noemi Santiago, Susan Haas, and Robert A. Baughman 1. Oral Delivery of Antigens . . . . . . . . 413

1.1. Introduction . . . . . . . . . . . . . . . . . . . 413 1.2. Approaches for Orallmmunization . . . . . . . 415

2. Novel Adjuvants and Vehicles for Oral Immunization 421 2.1. Surfactant-Based Vehicles ... 421 2.2. Microorganism-Based Delivery. . . 422 2.3. Microspheres . . . . . . . . . . . . 426

3. Proteinoid-Based Oral Antigen Delivery 426 3.1. Review of the Technology ..... 426 3.2. Oral Immunization with Flu Antigens 427 3.3. Ovalbumin as a Model Antigen . 429 References . . . . . . . . . . . . . . . . . 433

Chapter 18 Design and Production of Single-Immunization Vaccines Using Polylactide Polyglycolide Microsphere Systems Jeffrey L. Cleland 1. Introduction ...................... 439

1.1. Rationale for Single-Immunization Vaccines. . . 439 1.2. Potential Single-Immunization Vaccine Formulations 440

2. Predevelopment Studies for Design of in Vivo Release Profile. 445 3. Relationship Between Polymer Properties and Vaccine Design 447 4. Stability Constraints on Subunit Vaccines in Polylactide Microspheres 449 5. Manufacturing Hurdles for Polylactide Microsphere-Based Vaccines 451

5.1. Aseptic Manufacturing and Terminal Sterilization . . . . . . . 451 5.2. Scaleup, Process Integration, and Solvent Handling Concerns 453

6. Regulatory and Toxicology Issues . . . . . . . . . . 455 6.1. Residual Solvent Concerns . . . . . . . . . . . . . . . . . . . 456 6.2. Histology Studies of Poly lac tide Formulations. . . . . . . . . . 457

7. Overview of Major Issues in Design of Polylactide Vaccine Formulations . 458 8. Future Directions in the Development of Single-Shot Subunit Vaccines . 459

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459

Chapter 19 Nanoparticles as Adjuvants for Vaccines Jorg Kreuter I. Introduction .............. . 2. Production of Nanoparticles . . . . . . . . . . . . . . .

2.1. Polymerization in the Presence of the Immunogen. 2.2. Polymerization in the Absence of the Immunogen 2.3. Nanoparticles Produced in an Organic Phase ....

463 464 464 464 465

Contents xxvii

3. Characterization of Nanoparticles ........................ . 4. Influence of Particle Size and Hydrophobicity on the

Adjuvant Effect of Nanoparticles . . . . . . . . . . . 5. Antibody Response and Protection Induced with Vaccines

Containing PMMA Nanoparticles . . . . . . . . . 6. Body Distribution, Elimination, and Toxicological

Reactivity of PMMA Adjuvants 7. Summary .

References

Chapter 20 Water-Soluble Phosphazene Polymers for Parenteral and Mucosal Vaccine Delivery

Lendon G. Payne, Sharon A. Jenkins, Alexander Andrianov, and Bryan E. Roberts

465

466

467

469 470 471

1. Introduction ................... 473 2. Microsphere Formulation and Characterization. 476

2.1. Microsphere Generation ..... 476 2.2. Characterization of Microspheres . 478 2.3. Antigen Release 478

3. Immunogenicity Studies . . . 482 3.1. Parenteral Immunization 482 3.2. Mucosal Immunization . 490 3.3. The Role ofPolyphosphazene in Vaccine Delivery 491

4. Summary . 492 References 492

Chapter 21 Monopbospboryl Lipid A as an Adjuvant: Past Experiences and New Directions J. Terry Ulrich and Kent R. Myers 1. Introduction ................... 495

1.1. Historical Perspective . . . . . . . . . . . . 495 1.2. Rationale for Using MPL ® as an Adjuvant . 496

2. Characteristics of MPL ® as a Product 497 2.1. Manufacturing, Chemistry, and Quality Control . . 497 2.2. Stability ofMPL ®-Considerations for Formulation 501

3. Methods of Using MPL ® as an Adjuvant 502 3.1. Aqueous (Nonparticulate) Vehicles. . . . . . . . . . 503 3.2. Particulate Vehicles . . . . . . . . . . . . . . . . . . . . 503

4. Past Experiences-The Effectiveness of MPL ® as an Adjuvant 505 4.1. Preclinical Experience .......... 505 4.2. Clinical Experiences ........... 513 4.3. Modes of Action of MPL ® as an Adjuvant 516

5. New Directions. . . . . . . 517 6. Summary and Conclusions 518

References . . . . . . . . . 518

xxviii Contents

Chapter 22 Structural and Immunological Characterization of the Vaccine Adjuvant QS-21 Charlotte Read Kensil, Jia-Yan Wu, and Seall Soltysik 1. Introduction .......................... 525

1.1. Historical Use of Quillaja Saponins as Adjuvants . . . . 525 1.2. Discovery of Diversity of Quillaja Saponaria Adjuvants 526 1.3. Stimulon™-QS-21 Adjuvant 526

2. Chemical and Physical Properties 526 2.l. Structure . . . . . . . . 526 2.2. Amphipathic Character . . . 527

3. Manufacturing . . . . . . . . . . 527 3.l. Manufacturing and Quality Control 527 3.2. Formulation . 528 3.3. Stability . . . . . . 529

4. Adjuvant Activity ... 530 4.1. Antibody Response 530 4.2. Cell-Mediated Immune Response 533 4.3. Adjuvant Effect in Other Species . 533

5. Mechanism of Action ......... 534 5.1. Effect of Accessory and Effector Cell Depletion on CTL and Antibody

Response .......................... 534 5.2. Cytokine Profile Induced by QS-21-Adjuvanted Vaccines. 535 5.3. StructurelFunction Studies . 537

6. Clinical Experience with QS-21 . 538 7. Summary and Future Directions . 539

References . . . . . . . . . . . . 539

Chapter 23 A Novel Generation of Viral Vaccines Based on the ISCOM Matrix G. F. Rimme/zwaan and A. D. M. E. Osterhaus 1. Introduction .................... . 2. The ISCOM Structure ............... .

2.1. Components, Chemical and Physical Properties 2.2. Construction of ISCOMs . . . . . . . . . . .

3. Induction of B- and T-Cell Responses by ISCOMs 3.l. Induction of B-Cell Responses . . . . . . . . 3.2. Induction ofT-Cell Responses ....... .

4. Viral Vaccines Based on ISCOMs: Examples and State of the Art 4.1. Influenza Viruses 4.2. Paramyxoviruses 4.3. Herpesviruses . 4.4. Rhabdoviruses . 4.5. Hepadnaviruses 4.6. Retroviruses ..

5. General Considerations

543 543 543 545 545 545 546 547 547 550 550 551 551 552 552

Contents

5.1. Toxicological Aspects. 5.2. Mode of Administration.

6. Conclusions References

Chapter 24 Vaccine Adjuvants Based on Gamma Inulin Peter D. Cooper 1. Introduction ............... . 2. The Nature of Effective Vaccine Adjuvants 3. Involvement of Complement in Immune Defenses

3.1. Opsonic Effects of ACP Activation . . . . . 3.2. Immune Modulator Effects of CR Ligation

4. Complement Activators as Immune Modulators 5. Inulin as Complement Activator.

5.1. Chemistry ofInulin . . . . . 5.2. Formation of Gamma Inulin 5.3. Inulin and Complement ...

6. Gamma Inulin as Immune Modulator . 6.1. Gamma Inulin-The Product . . . 6.2. Vaccine Adjuvant Effects of Gamma Inulin 6.3. Effects of Gamma Inulin on Leukocyte-Cytokine Interactions

7. Algammulin-Composite of Alum and Gamma Inulin . 7.1. Algammulin-The Product . . . . . . . . . . . . . . . . . . 7.2. Vaccine Adjuvant Effects of Algammulin ......... . 7.3. Effects of Algammulin on Leukocyte-Cytokine Interactions

8. Safety Profile of Gamma Inulin-Based Vaccine Adjuvants 8.1. Gamma Inulin . . . . . . . . . . . . . 8.2. Algammulin . . . . . . . . . . . . . .

9. Comparison with Other Vaccine Adjuvants 10. Summary and Conclusions

References . . . . . . . . . . . . . . . . .

Chapter 25 A New Approach to Vaccine Adjuvants: Immunopotentiation by Intracellular T-Helper-Like Signals Transmitted by Loxoribine Michael G. Goodman

1. Introduction .......................... . 1.1. Discovery of Loxoribine and Its Analogues . . . . . . . . 1.2. Overview of Cell Types Activated and ImmunobiologicaJ

Actions Evoked by Loxoribine and Its Analogues 2. Amplification of Antibody Responses.

2.1. Cellular Mechanism of Action 2.2. T-Helper-Like Signals. 2.3. Animal Studies ... 2.4. Immunoprophylaxis . .

xxix

552 553 554 554

559 560 561 562 563 564 565 565 566 566 567 567 568 570 570 571 571 573 574 574 575 575 576 577

581 581

582 583 583 584 586 588

xxx Contents

2.5. Effects on Human Antibody Responses 3. Enhancement of T-Cell Responses. . . . . .

3.1. Augmentation of Cytolytic T-Cell Reactivity. 3.2. Antigen-Specific T-Cell Proliferation.

4. Role of Cytokines . . 5. Biochemical Studies . . . . . . . . . . . .

5.1. Transport System . . . . . . . . . . . 5.2. Loxoribine Binding Proteins: Binding Studies, Autoradiography 5.3. Interaction with Purine Salvage Pathway Enzymes 5.4. Protein Kinase C Pathway Independence

6. Preclinical Studies . . . . . . . . . . . . . . . . . . . 6.1. Advantageous Features Unique to Loxoribine .. 6.2. Dose Response, Vehicle, Route of Administration 6.3. Effects on Autoimmune-Prone Mice 6.4. Patent Status, Synthesis . . . . . . . . . . . . . 6.5. Safety Profile and Stability . . . . . . . . . . . 6.6. Activity Relative to Other Immunomodulators .

7. Clinical Trials ................... . 7.1. Phase I Trial in Advanced Cancer Patients . . . 7.2. Phase I Trial in Chronic Renal Failure Patients on Hemodialysis 7.3. Future Trials . . . . . .

8. Summary and Conclusions. References . . . . . . . . .

Chapter 26

589 591 591 591 592 593 593 593 594 596 596 596 597 599 599 601 601 603 603 603 604 605 605

Stearyl Tyrosine: An Organic Equivalent of Aluminum-Based Immunoadjuvants Christopher Penney 1. Introduction ........ 611 2. Why Replace Aluminum? . 612 3. Stearyl Tyrosine: Overview 612

3.1. Synthesis ....... 614 3.2. Toxicity . . . . . . . . 615

4. Adjuvanticity with Bacterial Vaccines. 615 5. Adjuvanticity with Viral Vaccines 617 6. Other Applications. . . . . . . 619 7. Analogues of Stearyl Tyrosine . 620 7. Conclusions 622

References 623

Chapter 27 Cytokines as Vaccine Adjuvants: Current Status and Potential Applications Penny Dong, Carsten Brunn, and Rodney J. Y. Ho 1. Introduction ..................... 625

1.1. The Role of Cytokines in Antigen Presentation 626 1.2. The Role of Cytokines in T-Cell Growth . 627 1.3. The Role of Cytokines in B-Cell Growth 627

Contents xxxi

2. Application ofCytokines in Modulating Antigen Presentation 628 2.1. Cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . 628 2.2. Cytokine Induction by Other Adjuvants . . . . . . . . . . 629

3. Application of Cytokines in Modulating T-Cell Growth to Enhance CellularImmune Response .. . . . . . . . . . . . . . . . . . . . . 631

4. Application of Cytokines in Modulating B-Cell Growth to Enhance Humoral Immune Response . . . . . . . . . . . . . . . . . . . . . . . . . . 632

5. Cytokine Fragments as Vaccine Adjuvants . . . . . . . . . . . . . 633 6. Local Delivery of Cytokines and the Sequence of Administration. 634 7. Conclusions 638

References 639

Chapter 28 Cytokines as Immunological Adjuvants

Andrew W Heath 1. Introduction ..... . 2. Production of Cytokines 3. Patent Protection .... 4. A Brief Summary of Cytokine Adjuvanticity

4.1. Interleukin-l . 4.2. Interleukin-2 .............. . 4.3. Interferon-y . . . . . . . . . . . . . . .

5. Means of Improving Adjuvant Effects of Cytokines 6. Areas of Particular Potential for Cytokine Adjuvants .

6.1. Immunodeficiency ........ . 6.2. Direction of the Immune Response .

7. Conclusions References

Chapter 29 Cytokine-Containing Liposomes as Adjuvants for Subunit Vaccines Lawrence B. Lachman. Li-Chen N. Shih. Xiao-Mei Rao. Stephen E. Ullrich. and Jeffrey L. Cleland

645 646 646 647 647 648 649 649 650 650 652 654 655

1. Introduction .............. 659 2. Cytokines as Adjuvants .. . . . . . . 659 3. Presentation of Antigen by Liposomes 660 4. Dehydration-Rehydration Vesicles (DRVs) Containing Cytokines . 662

4.1. Preparation of DRVs . . . . . . . . . . . . . . . . . . . . . . 662 4.2. Trapping Capacity of DRVs. . . . . . . . . . . . . . . . . . . 662 4.3. Slow Release of the Trapped Cytokines and HIV-l MN rgp 120 in the DRVs 663 4.4. Localization of Trapped Proteins . . . . . . . . . . . . . . . . . . . . . . . 664

5. Immunization of Mice with DRVs Containing Cytokines and HIV-I MN rgpl20 665 6. Possible Mechanisms of Action ofDRVs . . . . . 667 7. Future Directions for Cytokine-Containing DRV s 669

References . . . . . . . . . . . . . . . . . . . . . 669

xxxii Contents

Chapter 30 Haemophilus inJluenzae Type b Conjugate Vaccines Peter J. Kniskern, Stephen Marburg, and Ronald W Ellis 1. Introduction ..................... . 673 2. Rational Conjugate Design, Chemistry, and Process Control . 675

2.l. Outline of the Processes. . . . . . . . . . . . . . . . . . 675 2.2. Process Design and Quantitative Aspects of Covalency . 679 2.3. Characterization of Starting Materials and Process Intermediates 680 204. Separation of Un conjugated Polysaccharide . . . . . . . . . . 682

3. Characterization of Bulk Conjugates and Final Container Vaccines 682 4. Immunogenicity and Efficacy . . . . . . . . . . . . . . . . . . . . 687 5. Lessons for the Future . . . . . . . . . . . . . . . . . . . . . . . . 689

5.1. The Implications of Carrier Choice and of Size of Starting Ps . 689 5.2. The Importance of Rigorous Process Control and Analytical Testing of the

Process Intermediates, Final Products, and Formulated Vaccines 690 6. Summary. 690

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 691

Chapter 31 Pneumococcal Conjugate Vaccines Ronald Eby 1. Introduction .......... . 695

1.1. Epidemiology of Pneumococcal Disease 695 1.2. Current Licensed Pneumococcal Vaccines 697 1.3. The Need for Pneumococcal Conjugate Vaccines 697

2. Pneumococcal Conjugate Vaccines 698 2.1. Chemical Structures . 698 2.2. Preclinical Analysis . 703 2.3. Clinical Trial Results 706

3. Conclusions 713 References . . . . . . . . 714

Chapter 32 Lyme Vaccine Enhancement: N-Terminal Acylation ofa Protein Antigen and Inclusion of a Saponin Adjuvant Richard T. Coughlin, Jianneng Ma, and Daniel E. Cox 1. Background ..................... 719

1.1. Lyme Disease, Incidence, Transmission, and Symptoms. 719 1.2. The Biochemistry of Bacterial Lipoprotein Biosynthesis 720 1.3. Comparison of Acylated and Nonacylated Forms as Immunogens 721 104. Attempts at Synthetic Generation of Lipoproteins . . . . . . . 722

2. Product Description of Borrelia Burgdorferi Lipoprotein Vaccines 725 2.1. E. coli Expression and Purification 725 2.2. Vaccine Safety . . . . . . . . . . . . . . . . . . . . . . . . . . 727

Contents xxxiii

3. Vaccine Evaluation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 727 3.1. What Is the Nature of Vaccine-Induced Protection? . . . . . . . . . . .. 727 3.2. Differences in Immunoprotective Antibodies Induced by Acylated versus

Nonacylated Outer Surface Proteins . . . . . . . . . . . . . . . . . . .. 729 3.3. Cross Neutralization of Genetically and Geographically Divergent Borrelia 729

4. Future Directions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 730 4.1. Progress of Clinical Trials in High-Incidence Areas of the United States 730 4.2 Social Behavior as a Factor in the Control of Lyme Disease 730 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 731

Chapter 33 Vaccine Research and Development for the Prevention of Filarial Nematode Infections Robert B. Grieve, Nancy Wisnewski, Glenn R. Frank, and Cynthia A. Tripp

1. Introduction ............................. 737 2. Evidence for Protective Immunity in Filarial Nematode Infections 738

2.1. Existence of Naturally Acquired Protective Immunity. 739 2.2. Evidence of Concomitant Immunity in Filariasis 743 2.3. Experimental Immunization Studies . . . . . . . . . . 745

3. Characteristics of the Ideal Vaccine . . . . . . . . . . . . . 749 4. Dirofilaria immitis: A Vaccine in Animals and a Model for Human Filariases. 750 5. Identification of Candidate Antigens ....... . . . . . . . . . . . . . .. 751

5.1. Dirofilaria immitis Antigens Recognized by Antibodies in Immune Dog Sera 752 5.2. Lymphatic Filariasis. 753 5.3. Onchocerciasis ......................... 753 5.4. Microfilaria . . . . . . . . . . . . . . . . . . . . . . . . . . . 754

6. Cloning, Characterization, and Expression of Candidate Antigens . 754 7. Summary. 759

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 760

Chapter 34 Retrovirus and Retrotransposon Particles as Antigen Presentation and Delivery Systems Sally E. Adnms and Alan J. Kingsman 1. Introduction .................... 769 2. Presentation ofImmunogens as Hybrid Ty-VLPs . 770

2.1. The Yeast Ty Element . . . . . . . . . . . . . 770 2.2. Hybrid Ty-VLPs. . . . . . . . . . . . . . . . 772 2.3. Immune Responses Elicited by Hybrid VLPs 772

3. Presentation of Antigens as Virus-Derived Particles 778 3.1. The Construction of Hybrid Retroviral Cores . 778 3.2. Expression of Authentic Glycoproteins Using VDPs 780

4. Conclusions 782 References . . . . . . . . . . . . . . . . . . . . . . . . . 782

xxxiv Contents

Chapter 35 Rationale and Approaches to Constructing Preerythrocytic Malaria Vaccines Stephen L. Hoffman and John B. Sacci, Jr. 1. Irradiated Sporozoite Vaccination as a Model for Malaria Vaccine Development 7 S7 2. Preventing Sporozoite Invasion of Hepatocytes . . . . . . . . . . . . 7SS 3. Attacking the Infected Hepatocyte. . . . . . . . . . . . . . . . . . . 791

3.1. CDS+ T-Cell-Dependent Irradiated Sporozoite Vaccine-Induced Protective Immunity ....................... 791

3.2. Adoptive Transfer of CDS+ CTLs against the CS Protein Are Protective. 792 3.3. Immunization with CS Protein Vaccines Induces CDS+ T-Cell-Dependent

Partial Protection in Rodent Malaria Model Systems ........ 792 3.4. Induction of CTLs against the P. Jalciparum CS Protein in Humans 793 3.5. Identification of Sporozoite Surface Protein 2 as a Target of

Vaccine-Induced CDS+ Protective CTLs . . . . . . . . . . . . . . 794 3.6. CD4+ CTLs against the CS Protein Mediate Protective Immunity. 794 3.7. Other Parasite Antigen Targets in Infected Hepatocytes . . . . . . 795 3.S. Interferon-y and Other Cytokines . . . . . . . . . . . . . . . . . . 795

4. The Future: Inducing Multiple Immune Responses against Multiple Targets 796 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 797

Chapter 36 The MAP System: A Flexible and Unambiguous Vaccine Design of Branched Peptides Bernardetta Nardelli and James P. Tam 1. Introduction ....... . 2. MAP Design and Synthesis 3. MAP Antigenicity . . . . . 4. MAP Immunogenicity . . .

4.1. Production of a High-Titered Antibody Response 4.2. Reactivity of the Elicited Antisera against the Cognate Proteins. 4.3. Correlation MAP Structure and Immunogenicity ....... . 4.4. Enhanced Immunogenicity of MAPs Containing B- and T-Cell

Epitopes, and Protection Mediated by the Immune Sera . . . . . 4.5. Generation of Long-Term Antibody Responses ........ . 4.6. Capacity of Overcoming MHC-Associated Nonresponsiveness . 5. Lipidated MAP Vaccines

6. Concluding Remarks. References . . . . . . . . .

Chapter 37 Design of Experimental Synthetic Peptide Immunogens for Prevention of HIV-l and HTLV-I Retroviral Infections Mary Kate Hart, Thomas J. Palker; and Barton F. Haynes

S03 S04 S06 S06 S07 S07 SOS

SOS SIO SIl SIl S14 S16

1. Introduction .................................... 821

Contents xxxv

2. Synthetic Peptide Approach for HIV-I ........ 822 2.1. Peptide Synthesis and Purification . . . . . . . . 823 2.2. Design of HI V-I Th-B Hybrid Synthetic Peptides 823 2.3. Induction of Neutralizing Antibody Responses by Th-B

Peptide Immunogens . . . . . . . . . . . . . . . . . . . 825 2.4. Rhesus Monkey Studies ................. 826 2.5. Peptide-Induced Anti-HIV-l Responses in Chimpanzees 828 2.6. Effect of gp41 Fusogenic Domain (F) on Immune Response 829 2.7. Peptide Induction of MHC Class I Molecule-Restricted Anti -HIV-I CTLs 831

3. Simian Immunodeficiency Virus (SIV) env Synthetic Peptides Induce SIV-Specific CD8+ CTLs in Rhesus Monkeys 833

4. HTLV-I . . . . . . . . . . . . . . . . . . . . . 834 4.1. HTLV-I Pathogenesis and Epidemiology 834 4.2. Feasibility of an HTLV-I Vaccine. . . . . 834 4.3. Envelope Sequence Divergence among HTLV-I Strains 835 4.4. Vaccine-Induced Protection from HTLV-I Challenge . 835 4.5. Synthetic Peptide Approaches to HTLV-I Vaccine Development 836

5. Summary and Future Directions . 838 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 839

Chapter 38

Design and Testing of Peptide-Based Cytotoxic T-Cell-Mediated Immunotherapeutics to 1reat Infectious Diseases and Cancer

Robert W. Chesnut. Alessandro Sette. Esteban Celis. Peggy Wentworth. Ralph T. Kubo. Jeff Alexander, Glenn Ishioka. Antonella Vitiello. and Howard M. Grey

1. Background .................................... 847 1.1. The Role of Cytotoxic T Cells in the Control and Elimination of

Infectious Diseases and Cancer. . . . . . . . . . . . . . . . . . 847 1.2. The Processing and Presentation of Antigen to CD8+ Cytotoxic T Cells 849 1.3. Summary of Evidence Showing That Synthetic Peptides

Can Serve as Immunogens 850 2. Selection of Antigenic Peptides . . . . . 851

2.1. Introduction . . . . . . . . . . . . . 851 2.2. Definition ofMHC-Binding Motifs 851 2.3. Validation of the MHC-Binding Motifs. 855 2.4. Screening Vaccine Candidate Peptides for MHC Binding and in Vitro and

in Vi vo CTL Acti vation . 856 3. Therapeutic Applications ... 859

3.1. Background . . . . . . . . 859 3.2. Adoptive Cellular Therapy 859 3.3. In Vivo Injectable Immunotherapeutics 861

4. Summary and Perspectives 866 References . . . . . . . . . . . . . . . . . . 867

xxxvi

Chapter 39 Development of Active Specific Immunotherapeutic Agents Based on Cancer-Associated Mucins John Samuel and B. Michael Longenecker 1. Introduction ............. . 2. Immune Responses Relevant to Cancer Rejection 3. Target Antigens for ASI .. . . . . . . . . . . 4. Cancer-Associated Mucins as Target Antigens 5. Immune Responses to Carbohydrate Epitopes 6. Immune Responses to Mucin Core Peptides 7. Pharmaceutical Formulation of ASI Agents . 8. Summary.

References . . . . . . . . . . . . . . . . . .

Chapter 40 Synthetic Peptide Vaccines for Schistosomiasis Donald A. Ham, Sandra R. Reynolds, Silas Chikunguwo, Steve Furlong, and Charles Dahl

Contents

875 876 877 878 880 882 885 885 886

1. Background ............... 891 2. Targets of Immune Response ...... 892 3. Selection of Candidate Vaccine Antigens 893

3.1. Selection of B- and T-Cell Peptide Epitopes for Sm23 and TPI 894 3.2. Vaccination of Mice with TPI and Sm23 MAPs . . . 898 3.3. Immunobiology of TPI and Sm23 MAP Vaccination 900

4. Conclusions 901 References . . . . . . . . . . . . . . . . . . . . . . . . . 902

Chapter 41 Synthetic Hormone/Growth Factor Subunit Vaccine with Application to Antifertility and Cancer Laura L. Snyder; David V. Woo, Pierre L. Triozzi, and Vernon C. Stevens 1. Introduction .............................. 907 2. Special Considerations in Vaccine Development for Fertility Control 908 3. Immunological Fertility Control. . . 909

3.1. Hormones as Target Antigens . . . . . . . . . . . . . 910 3.2. Human Chorionic Gonadotropin . . . . . . . . . . . 910

4. Development of an Antifertility Vaccine Based on Human Chorionic Gonadotropin . . . . . 911

4.1. Peptide Immunogen Selection ...... 911 4.2. Carrier Selection ............. 912 4.3. Coupling and Ratio of Peptide to Carrier. 912 4.4. Adjuvant and Vehicle Selection. . . . . . 913

5. Preclinical Vaccine Experience in Antifertility 913 6. Clinical Vaccine Experience in Antifertility. . 915

Contents

7. Application to Cancer: Immunological Control of Cancer . . . . . . . 8. Application to Cancer: Tumor Expression of Chorionic Gonadotropin. 9. Application to Cancer: Clinical Vaccine Experience

10. The Future of the hCGpCTP37-DT Vaccine 10.1. Future Development for Antifertility 10.2. Future Development for Cancer. References . . . . . . . . . . . . . . .

Index . ....

xxxvii

916 918 921 923 923 924 925

931

List of Abbreviations

Ab ACP AFV Ag AIDS APCs ASI ATL

P2M BCG BHV BSA BuA2

CBER CDC CDER cm CDV CFA CFR CG c.1. CMI CMV CRM197

CSF CT CTB CTLs CTTH CVID CWS

antibody alternative complement pathway antiferility vaccine antigen acquired immunodeficiency syndrome antigen-presenting cells active specific immunity (immunotherapy) adult T-celileukemiallymphoma

P2-microglobulin bacillus Calmette-Guerin bovine herpesvirus bovine serum albumin l,4-butanediamine

Center for Biologics Evaluation and Research Centers for Disease Control Center for Drugs Evaluation and Research carbonyl diimidazole canine distemper virus complete Freund's adjuvant Code of Federal Regulations chorionic gonadotropin confidence interval cell-mediated immunity cytomegalovirus genetic mutant of diptheria toxin colony-stimulating factor cholera toxin B subunit of cholera toxin cytotoxic T lymphocytes N-benzyloxycarbonyl-L-tyrosyl-L-tyrosine hexyl ester common variable immunodeficiency disease cell wall skeleton

xxxix

xl

D DEC DMF DNP DNP-Pn DP DPT DT

DTH DTP

8BrGuo 8MGuo EBV ED50 EDAC EDTA ELA ELISA ER E:T EVAc EYPC

F(ab) FACS FCS FDA FHA FlTC FlV FMDV FSH F-MuLV

y-In GALT gB G-CSF GM-CSF GMP GnRH GPC GXM

diptheria toxoid diethylcarbamazine dimethylformamide dinitrophenol dinitrophenylated pneumococcus antigen degree of polarization diphtheria-pertussis-tetanus diphtheria toxoid diphtheria-tetanus delayed-type hypersensitivity diphtheria-tetanus-pertussis

8-bromoguanosine 8-mercaptoguanosine Epstein-Barr virus 50% effective dose l-ethyl-3-(3-dimethylaminopropyl) carbodiimide ethylenediaminetetraacetic acid establishment license application enzyme-linked immunosorbent assay endoplasmic reticulum effector-to-target ratio ethylene-vinyl acetate egg yolk phosphatidylcholine

antigen-binding fragment fluorescence-activated cell sorter fetal calf serum Food and Drug Administration filamentous hemagglutinin fluorescein isothiocyanate feline immunodeficiency virus foot-and-mouth disease virus follicle-stimulating hormone Friend murine leukemic helper virus

gamma-inulin gut-associated lymphoid tissue glycoprotein B granulocyte colony-stimulating factor granulocyte-macrophage colony-stimulating factor Good Manufacturing Practices gonadotropin-releasing hormone gel permeation chromatography glucuronoxylomannan



HA HAV HbOC HBsAg HBV hCG HCI HCV HEPES HGG Hib HIV HLA HLB HMO hPIV-I HPLC HPV HRP HSP HSV HTLV

ID50

IEF IFA IFN Ig IL i.m. IND i.p. IPV IRIVs ISCOMs i.v.

kD KLH

LAL LCMV LD50

LH LHRH

hemagglutinin hepatitis A virus Lederle-Praxis Hib conjugate hepatitis B surface antigen hepatitis B virus human chorionic gonadotropin hydrochloric acid hepatitis C virus N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid human gamma globulin Haemophilus inflllen~ae type b human immunodeficiency virus histocompatibility leukocyte antigen hydrophile-lipophile balance health maintenance organization human parainfluenza virus type I high-pressure liquid chromatography human papilloma virus horseradish peroxidase heat-shock protein herpes simplex virus human T-cellieukemiallymphotropic virus

50% infective dose or 50% inhibiting dose isoelectric focusing incomplete Freund's adjuvant interferon immunoglobulin interleukin intramuscular investigational new drug intraperitoneal inactivated polio vaccine immunopotentiating reconstituted influenza virosomes immune-stimulating complexes intravenous

kilodaltons keyhole limpet hemocyanin

Lim II Ills amoebocyte lysate lymphocyte choriomeningitis virus 50% lethal dose luteinizing honnone luteinizing hormone-releasing honnone

xli

xlii

LPS LT

MAb MAF MAP M-CSF MDP MELs MHC mIgA MLA MMR MPL MPL-A MTP-PE

NA NIAID NK NMR

OD OMPC OPV Osp Ova OVA

P PA PAGE PBLs PBMCs PBS PCPP PE PFCs PGA PK-C PLA

PLGA PLL

lipopolysaccharide lymphotoxin heat-labile toxin of E. coli

monoclonal antibody macrophage activation factor multiple antigen peptide macrophage colony-stimulating factor muramyl dipeptide microencapsulated liposomes major histocompatibility complex membrane-bound IgA 4'-monophosphoryllipid A measles-mumps-rubella monophosphoryllipid A monophosphoryl lipid A muramyl tripeptide phosphatidylethanolamine

neuraminidase National Institute of Allergy and Infectious Diseases natural killer nuclear magnetic resonance

optical density


outer membrane protein complex from Neisseria meningitidis B oral poliovirus vaccine outer surface protein ovalbumin ovalbumin

probability protective antigen polyacrylamide gel electrophoresis peripheral blood lymphocytes peripheral blood mononuclear cells phosphate-buffered saline poly[ die carboxylatophenoxy )phosphazene] phosphatidylethanolamine plaque-forming cells polyglycolic acid protein kinase C polylactic acid product license application polylactic coglycolic acid (poly-Iactide-co-glycolide) poly-L-Iysine


PMMA p.o. POE POP

polymethyl methacrylate per oral or oral polyoxyethylene polyoxypropylene

Pr protein PRP polyribosyl ribitol phosphate PRP-D Connaught Rib conjugate PRP-OMPC Merck Rib conjugate PRP-T Pasteur Merieux Rib conjugate Ps capsular polysaccharide PYA polyvinyl alcohol PVP polyvinylpyrrolidone PZC point of zero charge

r recombinant (e.g., rIFN-y) R receptor (e.g., IL-2R) RBCs red blood cells RIA radioimmunoassay RP-RPLC reverse phase RPLC RSV respiratory syncytial virus RT reverse transcriptase

7a8oGuo 7 -allyl-8-oxoguanosine 7m8oGuo 7 -methyl-8-oxoguanosine SAF-! Syntex Adjuvant Formulation-! s.c. subcutaneous SCMRC S-carboxymethylhomocysteine SCMS S-carboxymethylcysteamine SD standard deviation SDS sodium dodecyl sulfate SE standard error SEB staphylococcal enterotoxin B SEC size-exclusion chromatography SEM standard error of the mean SFCs spot-forming cells slgA secretory IgA SIV simian immunodeficiency virus SLA soluble leishmania antigen ST stearyl tyrosine STn sialyl Tn

('/2 half-life, half-time Ta Tamplifier TAA tumor-associated antigen TAP transporter protein

xliii

xliv

TCA TCR TO TOM TF TGF TGR Th TI TIL TLC TNF TNP-OVA TPE TPI Ts TSP/HAM IT

uv

VOPs VLPs VSV

WHH WHO

trichloroacetic acid T-cell receptor thymus-dependent trehalose dimycolate Thomsen-Friedenreich transforming growth factor trans-Golgi reticulum T-helper cell (e.g., Th 1) thymus-independent tumor-infiltrating lymphocyte thin-layer chromatography tumor necrosis factor trinitrophenyl ovalbumin tropical pulmonary eosinophilia triose phosphate isomerase T suppressor


tropical spastic paraparesisIHTLV-associated myelopathy tetanus toxoid

ultraviolet

virus-deri ved particles viruslike particles vesicular stomatitis virus

width at half height World Health Organization
